Aim: To investigate the role of miR-564 in promoting the proliferation and differentiation of synovial mesenchymal stem cells (SMSCs) to chondrocytes. Methods: Third-generation SMSCs were used, and the experiments involved untreated SMSCs (control; Group A), SMSCs transfected with Hsa-miR-564 inhibitor NC (inhibitor blank; Group B), and SMSCs transfected with Hsa-miR-564 inhibitor (Group C). The expression of miR-564 in SMSCs was determined by real-time quantitative polymerase chain reaction. The SMSCs were induced to form cartilage for 3 weeks. The morphology of the induced chondrocytes was observed by hematoxylin and eosin and toluidine blue staining and cell viability recorded. Chondrocyte differentiation of SMSCs related to genes and proteins (COL2A1, Aggrecan, SOX9, transforming growth factor beta 1 [TGF-β1], and Smad4) was assessed. The chondrogenic effect of miR-564 was examined after blocking the target gene TGF-β1. Results: The morphology and characteristics of the induced cells were consistent with those of chondrocytes. The cell proliferative rate of Group C (miR-564 downregulation) was significantly higher than that of other groups. The expression of genes and proteins related to chondrocyte differentiation was significantly decreased in Group C. The relative expression of genes related to cartilage differentiation decreased after blocking TGF-β1. Conclusion: The downregulation mediated by miR-564 can promote the differentiation and proliferation of SMSCs into chondrocytes by targeting TGF-β1.